This proposal tests the T.I.G.E.R. platform (commercially referred to as the Ibis T5000) system in the ferret model of influenza and provides a high level of controlled detail not available from infected humans. The high resolution genotypic information derived from the mass spectrometrically-determined base compositions will provide unique capabilities with which to study and monitor the transmission and genetic drift of diverse influenza strains. This project will utilize the extensive experience of LRRI in animal models and aerosol technology in the ABSL-3 environment to test the Ibis T5000 system and associated influenza surveillance kit for sensitivity, specificity, early diagnosis capability, and aerosol exhalation in the ferret model of H3N2 and H5N1 influenza infection. Specific Aim #1: To identify the earliest time and respiratory secretion site of viral detection by TIGER, we will test secretions of ferrets infected by intranasal H3N2 and H5N1 strains in the ABSL-3 laboratory at LRRI. Sample RNA will be isolated by robot in the ABSL-3 and shipped to Ibis in California for RT-PCR and mass spectrometry analysis of amplicons on the T5000. To compare the sensitivity of T5000 system to standard viral culture and real-time RT-PCR we will test parallel aliquots of all samples. Specific Aim 2. To test the ability of TIGER to detect genetic drift through mutation, and shift through recombination and reassortment, we will infect ferrets with single strains (as part of Specific Aim #1) and with co-infections with H3N2 and H5N1 strains. During early infection and at necropsy secretions and organ samples will be analyzed for multiple genotypes detected by amplimers from six genomic segments. To test the in vivo emergence of resistant H5N1 strains during oseltamivir treatment we will monitor appearance and relative numbers of genotypically-defined resistant strains arising in vivo during 4 serial passages in ferrets. Specific Aim 3. To test the ability of TIGER to detect viral RNA in exhaled aerosols produced by infected ferrets throughout the course of disease, we will capture exhaled virus in our inhalation apparatus in the controlled environment of the bioBubble. We will compare the sensitivity of two aerosol sampling techniques to detect virus, high-volume air filters and liquid impingers and compare detection of exhaled virus with the standard quantitative real-time PCR and plaque assays. Finally we will compare the exhalation of different strains of H3N2 and H5N1 viruses. Relevance: The T5000 Diagnostic System provides a rapid (<8 hours) high-throughput sensitive diagnostic tool useful to direct early therapy in seasonal influenza virus infection, and increase the efficiency and efficacy of early therapy, isolation protocols and quarantine practices in pandemic influenza.